Peter J. Feilen

Multilayer capsules: a promising microencapsulation system for transplantation of pancreatic islets

In 1980, Lim and Sun introduced a microcapsule coated with an alginate/polylysine complex for encapsulation of pancreatic islets. Characteristic to this type of capsule is, that it consists of a plain membrane which is formed during a single procedural step. With such a simple process it is difficult to obtain instantly a membrane optimized with respect to all the properties requested for islet transplantation. To overcome these difficulties, it is recommended to build up the membrane in several consecutive steps, each optimized for a certain property. In this study, we have analysed such a multilayer microcapsule for the encapsulation of pancreatic islets. Therefore, empty and islet containing alginate beads were coated with alternating layers of polyethyleneimine, polyacrylacid or carboxymethylcellulose and alginate. By scanning electron microscopy the thickness of the covering multilayer-membrane was estimated to be less than 800 nm by comparison with an apparatus scale. Ellipsometric measurements showed that the membrane thickness is in the range of 145 nm. Neither the encapsulation procedure, nor the membrane-forming step did impede the stimulatory response of the islets. The encapsulation even lead to a significantly better stimulatory response of the encapsulated islets during week three and five of cell culture. Furthermore, the multilayer-membrane did not deteriorate the biocompatibility of the transplanted microcapsules, allowing an easy tuning of the molecular cut-off and the mechanical stability depending on the polycation-polyanion combination used. The multilayer membrane capsule has obvious advantages compared to a one-step encapsulation procedure. These beads guarantee a high biocompatibility, a precisely adjusted cut-off, an optimal insulin-response and high mechanical stability although the membrane is only 145 nm thick.

Size of pancreatic islets of Langerhans: a key parameter for viability after cryopreservation.

Abstract.Large amounts and excellent viabilities of pancreatic islets are prerequisites for recent advances in islet transplantation. Cryopreservation has been shown to enlarge transplanted cell mass, but has been accompanied by reduced viability. In this study rat pancreatic islets were differentiated into small (<200 µm), medium (200–400 µm) and large (>400 µm) categories and their susceptibilities to different freezing conditions were evaluated: concentration of cryoprotectant (0.7–3.1 M), equilibration (15 vs. 45 min, 22° C vs. on ice) and post-thaw removal of cryoprotectant (15 vs. 30 min, stepwise vs. one-step). The most prominent finding was a negative correlation between islet size and viability observed in non-frozen islets to a minor degree (r=-0.44) and significantly enhanced after cryopreservation (r<-0.8). The concentration of cryoprotectant showed the most significant influence on viability affecting small, medium and large islets. Different techniques of equilibration with the cryoprotectant resulted in significant changes of islet viability of medium islets, whereas small and large islets were unaffected. For different techniques of removal of the cryoprotectant, no significant influence on viabilities was found. We conclude that large islets represented a highly susceptible population concerning damage due to cryopreservation.

Biocompatibility of Alginates for Grafting: Impact of Alginate Molecular Weight

Optimising microencapsulation technology towards the effective clinical transplantation has created the need for highly biocompatible alginates. Therefore, in this study the biocompatibility of different beads prepared from alginates with varying average molecular weight was examined. In some experiments the beads were covered with a multilayer membrane surrounded by an alginate layer. First of all, we found that beads made of a lower weight average alginate elicted a much stronger fibrotic response compared to beads made of a higher weight average alginate (LV‐alginate > MV‐alginate). The results were confirmed by the observation that the extent of tissue fibrosis was significantly increased in multilayer capsules made of an alginate with a lower weight average (core and surface LV‐alginate, Mw 0.7–1*106 g/mol, viscosity of a 0.1% solution 1–2.5 mPa s− 1) compared to multilayer capsules made of an alginate with a higher weight average (core and surface MV‐alginate; Mw 1.2–1.3*106 g/mol, viscosity of a 0.1% solution 5–7 mPa s− 1). It should be stressed, that the pro‐fibrotic effect of the LV‐alginate alginate in the core was only partially reversed by a MV‐alginate on the surface of the multilayer capsules. On the basis of the raised data, it can be assumed that the molecular weight average of the alginates have an decisive effect on the biocompatibility. Therefore, it seems to be recommendable to reduce the low molecular weight fractions of the alginate during the purification process to improve the biocompatibility.

Magnetic separation of encapsulated islet cells labeled with superparamagnetic iron oxide nano particles

Islet cell transplantation is a promising option for the restoration of normal glucose homeostasis in patients with type 1 diabetes. Because graft volume is a crucial issue in islet transplantations for patients with diabetes, we evaluated a new method for increasing functional tissue yield in xenogeneic grafts of encapsulated islets. Islets were labeled with three different superparamagnetic iron oxide nano particles (SPIONs; dextran‐coated SPION, siloxane‐coated SPION, and heparin‐coated SPION). Magnetic separation was performed to separate encapsulated islets from the empty capsules, and cell viability and function were tested. Islets labeled with 1000 μg Fe/ml dextran‐coated SPIONs experienced a 69.9% reduction in graft volume, with a 33.2% loss of islet‐containing capsules. Islets labeled with 100 μg Fe/ml heparin‐coated SPIONs showed a 46.4% reduction in graft volume, with a 4.5% loss of capsules containing islets. No purification could be achieved using siloxane‐coated SPIONs due to its toxicity to the primary islets. SPION labeling of islets is useful for transplant purification during islet separation as well as in vivo imaging after transplantation. Furthermore, purification of encapsulated islets can also reduce the volume of the encapsulated islets without impairing their function by removing empty capsules.